In the human fetal testis, germ cells that have migrated to the genital ridges become enclosed within testicular cords by 8 wk of gestation. Most papers refer to all types of germ cells as being "gonocytes" or "prespermatogonia," giving the impression that they are identical. Detailed morphological studies, however, have suggested a heterogeneous population. We have used single, double, and triple immunohistochemistry to evaluate the differentiation of cells within fetal testes recovered during the first (7-9 wk) and second (14-19 wk) trimesters. In the first trimester, differentiation of Sertoli cells preceded the formation of testicular cords and the differentiation of interstitial (Leydig, peritubular myoid) cells. Immunostaining for CHK2, C-KIT, placental alkaline phosphatase, PCTAIRE-1, and MAGE-A4 revealed that the proportion of germ cells expressing each of these proteins was correlated with gestational age. Expression of the pluripotency marker OCT4 was restricted to a population of small, round germ cells. Three types of germ cell were identified, and we propose that these should be known as gonocytes (OCT4pos/C-KITpos/MAGE-A4neg), intermediate germ cells (OCT4low/neg/C-KITneg/MAGE-A4neg), and prespermatogonia (OCT4neg/C-KITneg/MAGE-A4pos). In the first trimester, most germ cells had a gonocyte phenotype; however, from 18 wk of gestation, prespermatogonia were the most abundant cell type. These data provide evidence for the functional differentiation of human testicular germ cells during the second trimester of pregnancy, and they argue against these germ cells being considered as a homogeneous population, as in rodents.

The thymus is essential for a functional immune system, because the thymic stroma uniquely supports T lymphocyte development. We have previously identified the epithelial progenitor population from which the thymus arises and demonstrated its ability to generate an organized functional thymus upon transplantation. These thymic epithelial progenitor cells (TEPC) are defined by surface determinants recognized by the mAbs MTS20 and MTS24, which were also recently shown to identify keratinocyte progenitor cells in the skin. However, the biochemical nature of the MTS20 and MTS24 determinants has remained unknown. Here we show, via expression profiling of fetal mouse TEPC and their differentiated progeny and subsequent analyses, that both MTS20 and MTS24 specifically bind an orphan protein of unknown function, Placenta-expressed transcript (Plet)-1. In the postgastrulation embryo, Plet-1 expression is highly restricted to the developing pharyngeal endoderm and mesonephros until day 11.5 of embryogenesis, consistent with the MTS20 and MTS24 staining pattern; both MTS20 and MTS24 specifically bind cell lines transfected with Plet-1; and antibodies to Plet-1 recapitulate MTS20/24 staining. In adult tissues, we demonstrate expression in a number of sites, including mammary and prostate epithelia and in the pancreas, where Plet-1 is specifically expressed by the major duct epithelium, providing a specific cell surface marker for this putative reservoir of pancreatic progenitor/stem cells. Plet-1 will thus provide an invaluable tool for genetic analysis of the lineage relationships and molecular mechanisms operating in the development, homeostasis, and injury in several organ/tissue systems. thymus development ͉ MTS24 ͉ endoderm ͉ mesonephros

Thymus function requires extensive cross-talk between developing T-cells and the thymic epithelium, which consists of cortical and medullary TEC. The transcription factor FOXN1 is the master regulator of TEC differentiation and function, and declining Foxn1 expression with age results in stereotypical thymic involution. Understanding of the dynamics of Foxn1 expression is, however, limited by a lack of single cell resolution data. We have generated a novel reporter of Foxn1 expression, Foxn1G, to monitor changes in Foxn1 expression during embryogenesis and involution. Our data reveal that early differentiation and maturation of cortical and medullary TEC coincides with precise sub-lineage-specific regulation of Foxn1 expression levels. We further show that initiation of thymic involution is associated with reduced cTEC functionality, and proportional expansion of FOXN1-negative TEC in both cortical and medullary sub-lineages. Cortex-specific down-regulation of Foxn1 between 1 and 3 months of age may therefore be a key driver of the early stages of age-related thymic involution.

Testicular cancer is more common in individuals with disorders of the male reproductive tract. It has been suggested that inappropriate exposure to estrogens during fetal life may have an impact on maturation of testicular germ cells that are the cells of origin of the majority of testis cancers. The aim of the present study was to establish whether human fetal germ cells (gonocytes) are a potential target of estrogen action. To address this issue, we used RT-PCR and immunohistochemistry to examine the pattern of expression of estrogen receptors (ER alpha, ER beta, and ER beta 2 variant) in human fetal testes at 12-19 wk gestation. ER alpha, mRNA, and protein were not detected in any of the fetal testes. In contrast, using an antibody directed against the hinge domain of ER beta expression was detected in multiple testicular nuclei. RT-PCR with primers specific for full-length wild-type ER beta (ER beta 1) or the ER beta 2 variant formed by splicing of an alternative eighth exon, was performed on whole-tissue extracts and materials recovered by laser capture and revealed that mRNAs for both isoforms were expressed. Immunohistochemistry with isotype-specific monoclonal antibodies showed that ER beta 1 was low/undetectable in gonocytes, whereas these cells expressed the highest levels of ER beta 2, compared with other testicular cell types. Both ER beta 1 and ER beta 2 were detected in some but not all Sertoli cells, peritubular cells, and other interstitial cells including those tentatively identified as Leydig cells. Our immunohistochemical results demonstrate that during the second trimester, some but not all somatic cells within the human fetal testis express wild-type ER beta (ER beta 1) protein and/or the variant isoform of ER beta (ER beta 2) that lacks amino acids essential for binding of estradiol. ER beta 2 protein was readily detectable in fetal gonocytes, whereas ER beta 1 was not. We did not detect expression of ER alpha. The expression of ER beta 2, a variant proposed act as a dominant negative receptor, might prevent estrogen action in gonocytes. We suggest that during this period of fetal life, estrogenic ligands are most likely to act on somatic cells that contain ER beta 1 protein.

A novel Drosophila melanogaster protein, termed inhibitor-t, that bears 41% sequence similarity to human protein phosphatase inhibitor-2 has been identified using human protein phosphatase 1 (PP1) in the yeast two hybrid system. Inhibitor-t mRNA is detected in adult males, larvae and pupae and the 184 amino acid thermostable protein located only in testis. The gene for inhibitor-t maps to cytological location 86F1 on the third chromosome. Bacterially expressed inhibitor-t specifically inhibits both mammalian and D. melanogaster PP1 catalytic subunits with an IC SH of approximately 200 nM. A motif -FEX I X P RK-, conserved between inhibitor-t, inhibitor-2 and its Saccharomyces cerevisiae homologue Glc8, is demonstrated to be required for binding to PP1.z 1998 Federation of European Biochemical Societies.

Background:Patients with acute myeloid leukemia (AML), myelodysplasia (MDS) or tyrosine kinase inhibitor resistant chronic myeloid leukemia (CML) who are unsuitable for consolidative allogeneic stem cell transplantation (alloSCT) have high relapse rates following chemotherapy. Wilms' tumor 1 (WT1) is highly expressed in the majority of acute myeloid leukemias (AML) and in many subtypes of myelodysplasia (MDS) as well as other hematological and solid tumors. WT1 is an intracellular antigen, which makes it difficult to target using current Chimeric Antigen Receptor (CAR)-T cell technologies. The use of genetically modified T cells expressing WT1-specific α/β T cell receptors can re-direct T cell specificity via the recognition of intracellular peptides presented by MHC molecules on the malignant cell surface. Phase I clinical trials of WT1-TCR gene-modified T cells have been conducted in the settings of relapsed disease and post-alloSCT and preliminary data suggests this treatment approach is safe and potentially clinically effective in these cohorts (Tawara et al. Blood. 2017;130(18):1985-94; Chapuis et al, Nat Med. 2019;25(7):1064-72). Methods:We report a phase I/II safety and dose escalation study evaluating WT1-TCR gene-modified autologous T cells in HLA-A*0201 positive patients with AML, MDS and CML, unsuitable for alloSCT (NCT02550535) (Fig 1A). Patient T cells were harvested by leucapheresis and transduced with a retroviral vector construct encoding the codon optimised variable and constant a and bchains of the human pWT126-specific TCR separated by a self-cleaving 2A sequence (Fig 1B). Bulk transduced T cells were analysed by flow cytometry (CD3, CD8 and Vb2.1) prior to infusion and at regular intervals post-infusion. A quantitative PCR assay was developed to identify WT1-TCR expressing T cells in the peripheral blood post infusion. Patients received minimal conditioning with fludarabine and methylprednisolone prior to transfer of transduced T cells. All subjects were followed for a minimum of 12 months or until death. Results:A total of 10 patients (6 AML, 3 MDS and 1 TKI- resistant CML) were recruited. The mean age was 71.3 years (range 64-75) and all had high risk disease (by cytogenetic or clinical criteria). All AML patients were in complete morphological remission at the time of trial entry, whilst MDS patients had ≤ 15% blasts on bone marrow examination. All 10 patients received the gene-modified T cells in dose escalation cohorts (seven patients received £2x107/kg and three patients received £1x108/kg bulk WT1 TCR transduced cells). No adverse events directly attributable to the investigational product were recorded apart from one possible cytokine release syndrome, which was managed without tociluzimab. Transferred T cells demonstrated in vivoproliferation commensurate with maintenance of functional capacity despite ex vivo manipulation (Fig 1C and 1D). The TCR-transduced T cells were detectable in all patients at 28 days and in 7 patients persisted throughout the study period (Fig 1E). All 6 AML patients were alive at last follow up (median 12 months; range 7-12.8 months). The 3 patients with MDS had a median survival of 3 months (range 2.1-3.96 months) post T cell infusion. 2 died from progressive disease and one from other causes. 2 patients discontinued the study early due to disease progression. Conclusions: This is the second reported phase I/II clinical trial of autologous WT1-TCR gene-modified T cells for treatment of AML and MDS in a high-risk cohort of patients not suitable for alloSCT. We have shown that the WT1-TCR T cells demonstrated a strong safety profile without detectable on-target, off-tumour toxicity and no severe adverse events in the ten patients treated. An important cause of treatment failure for adoptive cellular therapies is the lack of persistence of transferred T cells leading to loss of disease specific effects. We demonstrated that autologous WT1-TCR T cells proliferated in vivoand persisted for many months. Recent work within our group (in press) has shown that TCRs modified to include key framework residues, show increased TCR expression and functional improvement. These modifications could be incorporated into future studies to improve efficacy. This data supports the rationale for a larger, phase II trial of WT1-TCR T cells in myeloid malignancies in patients for whom alloSCT is not appropriate, in order to assess clinical efficacy. Figure 1 Disclosures Morris: Quell Therapeutics: Consultancy, Other: Scientific Founder,stock; Orchard Therapeutics: Consultancy. Qasim:CellMedica: Research Funding; Bellicum: Research Funding; UCLB: Other: revenue share eligibility; Autolus: Equity Ownership; Orchard Therapeutics: Equity Ownership; Servier: Research Funding. Mount:Gamma Delta Therapeutics: Employment. Inman:Cellmedica: Employment. Gunter:Cellmedica: Employment. Stauss:Cell Medica: Other: I have stock; Quell Therapeutics: Consultancy, Other: I have stock.

Under homeostatic conditions, dendritic cells (DCs) continuously patrol the intestinal lamina propria. Upon antigen encounter, DCs initiate C‐C motif chemokine receptor 7 (CCR7) expression and migrate into lymph nodes to direct T cell activation and differentiation. The mechanistic underpinnings of DC migration from the tissues to lymph nodes have been largely elucidated, contributing greatly to our understanding of DC functionality and intestinal immunity. In contrast, the molecular mechanisms allowing DCs to efficiently migrate through the complex extracellular matrix of the intestinal lamina propria prior to antigen encounter are still incompletely understood. Here we show that small intestinal murine CD11b+CD103+ DCs express Placenta‐expressed transcript 1 (Plet1), a glycophoshatidylinositol (GPI)‐anchored surface protein involved in migration of keratinocytes during wound healing. In the absence of Plet1, CD11b+CD103+ DCs display aberrant migratory behavior, and accumulate in the small intestine, independent of CCR7 responsiveness. RNA‐sequencing indicated involvement of Plet1 in extracellular matrix‐interactiveness, and subsequent in‐vitro migration assays revealed that Plet1 augments the ability of DCs to migrate through extracellular matrix containing environments. In conclusion, our findings reveal that expression of Plet1 facilitates homeostatic interstitial migration of small intestinal DCs.

Self-renewing stem cell populations are increasingly considered as resources for cell therapy and tools for drug discovery. Human pluripotent stem (hPS) cells in particular offer a virtually unlimited reservoir of homogeneous cells and can be differentiated toward diverse lineages. Many diseases show impairment in self-renewal or differentiation, abnormal lineage choice or other aberrant cell behavior in response to chemical or physical cues. To investigate these responses, there is a growing interest in the development of specific assays using hPS cells, artificial microenvironments and high content analysis. Several hurdles need to be overcome that can be grouped into three areas: (i) availability of robust, homogeneous, and consistent cell populations as a starting point; (ii) appropriate understanding and use of chemical and physical microenvironments; (iii) development of assays that dissect the complexity of cell populations in tissues while mirroring specific aspects of their behavior. Here we review recent progress in the culture of hPS cells and we detail the importance of the environment surrounding the cells with a focus on synthetic material and suitable high content analysis approaches. The technologies described, if properly combined, have the potential to create a paradigm shift in the way diseases are modeled and drug discovery is performed.